Ctr. for Excellence in Pulmonary Biology, Division of Pediatric Pulmonary, Asthma and Critical Care Medicine, Stanford Univ. Medical School Medicine, Stanford, CA 94305, USA.
Am J Physiol Lung Cell Mol Physiol. 2012 Feb 1;302(3):L352-9. doi: 10.1152/ajplung.00302.2011. Epub 2011 Nov 23.
Previously, we observed that hypoxia increases the expression of the β1-subunit (KCNMB1) of the calcium-sensitive potassium channel (BK(Ca)). Herein, we elucidate the mechanism whereby hypoxia increases KCNMB1 expression in human pulmonary artery smooth muscle cells (hPASMC). In response to hypoxia, the expression of both the transcription factor hypoxia-inducible factor 1-α (HIF-1α) and KCNMB1 are increased. Knockdown of HIF-1α using a shRNA plasmid blocked the hypoxic induction of KCNMB1 expression. Chromatin immunoprecipitation (ChIP) demonstrated HIF-1α binding to three discrete regions of the human KCNMB1 promoter known to contain hypoxia response elements (HREs). A KCNMB1 promoter reporter assay combined with site-directed mutagenesis identified two adjacent HREs located between -3,540 bp and -3,311 bp that are essential for the hypoxic induction of KCNMB1 promoter activity. Furthermore, additional ChIP assays demonstrated recruitment of the HIF-1α transcriptional coactivator, p300, to this same promoter region. Treatment of hPASMC with the histone deacetylase inhibitor, trichostatin, prolonged the increase in KCNMB1 observed with hypoxia, suggesting that alterations in chromatin remodeling function to limit the hypoxic induction of KCNMB1. Finally, KCNMB1 knockdown potentiated the hypoxia-induced increase in cytosolic calcium in hPASMC, highlighting the contribution of the β1-subunit in modulating vascular SMC tone in response to acute hypoxia. In conclusion, HIF-1α increases KCNMB1 expression in response to hypoxia in hPASMC by binding to two HREs located at -3,540 to -3,311 of the KCNMB1 promoter. We speculate that selective modulation of KCNMB1 expression may serve as a novel therapeutic approach to address diseases characterized by an increase in vascular tone.
先前，我们观察到缺氧会增加钙敏钾通道（BK(Ca)）β1 亚基（KCNMB1）的表达。在此，我们阐明了缺氧增加人肺动脉平滑肌细胞（hPASMC）中 KCNMB1 表达的机制。在缺氧的刺激下，转录因子缺氧诱导因子 1-α（HIF-1α）和 KCNMB1 的表达都增加。使用 shRNA 质粒敲低 HIF-1α 会阻止 KCNMB1 表达的缺氧诱导。染色质免疫沉淀（ChIP）显示 HIF-1α 与人类 KCNMB1 启动子上三个离散的区域结合，这些区域已知含有缺氧反应元件（HREs）。KCNMB1 启动子报告基因测定与定点诱变相结合，鉴定出位于 -3540bp 和 -3311bp 之间的两个相邻 HRE，对于 KCNMB1 启动子活性的缺氧诱导至关重要。此外，额外的 ChIP 测定表明 HIF-1α 转录共激活因子 p300 募集到同一启动子区域。用组蛋白去乙酰化酶抑制剂曲古抑菌素处理 hPASMC 可延长缺氧时观察到的 KCNMB1 增加，表明染色质重塑功能的改变限制了 KCNMB1 的缺氧诱导。最后，KCNMB1 敲低增强了 hPASMC 中缺氧诱导的细胞浆钙增加，突出了 β1 亚基在调节血管平滑肌细胞对急性缺氧的反应中的作用。总之，HIF-1α 通过与 KCNMB1 启动子上的-3540 至-3311 之间的两个 HRE 结合，在 hPASMC 中增加 KCNMB1 的表达以响应缺氧。我们推测，选择性调节 KCNMB1 的表达可能是解决血管张力增加的疾病的一种新的治疗方法。
